The precise location and the physiological function of the various adenine nucleotide binding site of the F1-ATP'ase will be explored with radioactive nucleotide analogues designed to react covalently with amino acid side chains at nucleotide binding sites in the enzyme. One such affinity label which inactivates the F1-ATP'ase irreversibly is p-fluoro-sulfonylbenzoyl-5'-adenosine. A synthesis has been worked out to label this analogue with 14C in the benzoyl moiety starting with 14C-p-aminobenzoic acid. Other affinity labels which will be used in this study are 7-chloro-4-nitrobenzo-2-oxa-l, 3-diazole, and the 2',3'-dialdehyde derivative of ATP. Experiments will be designed to label the adenine nucleotide regulatory site and the catalytic site(s) separately with the use of the affinity labels and appropriate competitive and non-competitive inhibitors of the enzyme. The subunit localization and the amino acid sequence around the labelled binding sites will be determined. In addition, cross-linking studies will be carried out to determine the proteins with which the F1-ATP'ase interacts in the mitochondrial inner membrane. A number of reagents which contain aryl azides and can be attached to surface lysine residues of proteins have been synthesized. When the aryl azido derivatives of the proteins are illuminated, the generated nitrene can react covalently with amino acid side chains in its environment with some of them residing on neighboring proteins. The aim of these studies is to determine the topology of the energy transducing proteins in the inner membrane of the mitochondrion.